The present application claims priority to German Application 198 34 430.9, filed Jul. 30, 1998.
The present invention relates to a recombinant vector, to a pharmaceutical composition containing the vector according to the invention, to the use of the vector according to the invention for treating tumor patients and to transduced eukaryotic cells.
So far benign and malign tumors have predominantly been treated either invasively, i.e. by surgical removal of the tumor, or conservatively, e.g. by administration of cytostatics or radiation of the organs affected, or by a combination of said methods. Great successes have already been achieved with these therapeutical possibilities because of permanently improving surgical techniques and a tremendous development in the field of cytostatics.
Nevertheless, the chances of success in the treatment of a tumor by said therapeutical methods vary considerably and are unpredictable. Moreover, each of the three methods has serious drawbacks. For instance, the surgical removal of a tumor and possibly the incision of healthy tissue greatly affects the patient because of the surgery itself. It is only in exceptional cases that the treatment with cytostatics and the radiation of tumors can be restricted to the target cells proper, so that it is almost unavoidable to subject healthy cells and healthy tissue to the treatment as well. A treatment with cytostatics means the inhibition of mitoses, which e.g. has the unpleasant side effect of alopecia. Both cytostatic treatment and radiation therapy may entail childlessness in patients of child-bearing and procreative age.
On account of the above-mentioned impairments attempts have already been made to transport therapeutics, in particular cytostatics, in a targeted manner to tumor cells and to have them internalized by said cells. An example thereof is the attempt to couple cytostatics to antibodies which bind to tumor cell-specific antigens. Despite the theoretical attractivity of such a proposal, this entails considerable difficulties, e.g. in obtaining sufficient amounts of antibodies, and also entails considerable risks, e.g. an immunological reaction to the foreign protein supplied.
A further approach is based on the knowledge about the role of transcription factors in the development of tumors. It is generally known that a few nucelar transcription factors serve to monitor the integrity of the cellular genome. When genomic DNA is damaged, said transcription factors will induce either a cell cycle arrest, which is required for repair, or apoptosis in the case of irreparable damage. Thus said transcription factors have an important tumor suppressor function. Many recent papers have been concerned with the transcription factor p53, in particular in connection with impaired p53 function in the development of cancer (Levine, 1997). The phenotype of p53xe2x88x92/xe2x88x92 mice, produced by means of xe2x80x9cgene targetingxe2x80x9d, demonstrates said connection: the absence of the protein results in an increased occurrence of spontaneous tumors (Donehower et al., 1992). Moreover, it could be demonstrated with the help of said mice that p53 plays a key role in the induction of apoptosis (Lowe et al., 1994).
In conventional tumor therapies, such as radiation or chemotherapy, it is this p53-mediated apoptosis induction that plays an important role (Lowe et al., 1993). On account of the accompanying resistance to cytostatics or radiation therapy, cancer types with mutated p53 (p53mut) have a poor prognosis most of the time. In humans mutations of or deletions in p53 are observed in 50-80% of all cancer types (Levine et al, 1991). They always regard the DNA binding domain and entail loss in the p53 transactivator function.
The genetic difference between p53 molecules in normal and transformed cells has recently been exploited for the selective elimination of tumor cells. An adenovirus mutant which can only propagate in p53-deficient cells destroyed transplanted human p53mut tumors selectively and efficiently in nude mice experiments (Bischoff et al., 1996). In humans, however, an already existing immunity to adenoviruses could turn out to be a great problem. Since the majority of the population has already been immunized by preceding adenovirus infections, it could be that most of the patients intended for therapy will eliminate the therapeutical virus before it can develop its desired killing potential. Therefore, adenoviral vectors are only suited to a limited degree for use in gene therapy.
Starting from said prior art, it has been the object of the present invention to provide ways and means to fight tumor cells in a targeted manner.
According to the invention, this object is achieved by a recombinant vector comprising:
(a) at least one first transcription cassette containing a sequence coding for a recombinase, a minimal promoter MP functionally linked thereto, a transcription factor binding site and optionally a polyadenylation sequence, wherein the minimal promoter MP depends on the activation by one or several transcription factors,
(b) at least one second transcription cassette containing a suicide gene, a promoter P functionally linked thereto and optionally a polyadenylation sequence;
(c) a 5xe2x80x2-flanked sequence and/or a 3xe2x80x2-flanked sequence, wherein the 5xe2x80x2-and/or 3xe2x80x2-flanked sequence contains a recombinase target sequence.
The vector according to the invention permits the targeted and selective elimination of tumor cells in that a suicide gene is introduced by the vector into the cells, said gene being immediately eliminated in healthy cells by expression of recombinase whereas it can be activated in tumor cells with inactive transcription factors and leads to cell death (i) by expression of the suicide protein, (ii) by transcription of antisense RNA or (iii) by production of cytopathogenic virus.
In the context of the present invention the term xe2x80x9cvectorxe2x80x9d means a linear or circular nucleic acid molecule which may consist of deoxyribonucleic acid and also of ribonucleic acid. Vectors suited for gene therapy, which may serve as starting material for the inventive vectors, are known in the prior art. Preferred vectors are vectors derived from viral or retroviral genomes because these can be packaged into viruses and can easily be introduced into cells by transduction. Vectors on a non-viral basis are also possible, but require further transfection measures. Suitable vectors would e.g. be fully synthetic vectors or vectors transduced by attenuated bacteria.
xe2x80x9cReplication competencexe2x80x9d means the ability of a vector to replicate in host cells. A very high replication competence with respect to mammalian cells is e.g. found in adenovirus, retroviruses, such as mouse leukemia virus MuLV, in particular Moloney mouse leukemia virus (MoMuLV). The invention generally comprises vectors with replication competence in eukaryotic cells. If the vectors are retroviruses, infectious retroviruses are preferred.
xe2x80x9cTranscription cassettesxe2x80x9d are nucleic acid units which apart from the sequence coding for a protein contain the necessary regulatory regions, e.g. promoter or minimal promoter with transcription factor binding site and polyadenylation sequences. The first transcription cassette may be located 5xe2x80x2 or 3xe2x80x2 relative to the second transcription cassette.
A xe2x80x9cminimal promoterxe2x80x9d is a natural or synthetic promoter or enhancer which can only activate the gene expression in the presence of a specific transcription factor. It contains at least one natural or synthetic transcription factor binding site.
A xe2x80x9ctranscription factor binding sitexe2x80x9d is a natural or synthetic nucleic acid sequence to which a transcription factor required for activating a minimal promoter can bind.
A xe2x80x9crecombinasexe2x80x9d is a natural or synthetic enzyme which recognizes and cuts specific target sequences and recombines the same with one another. Examples thereof are the Cre recombinase from the P1 coliphage (Sternberg and Hamilton, 1981) and the Flp recombinase from S. cerevisiae (Broach and Hicks, 1980).
xe2x80x9cA target sequencexe2x80x9d is a natural or synthetic sequence which can specifically be recognized by a recombinase. Examples thereof are the loxP sequences from the P1 coliphage (Gu et al., 1993; Sauer and Henderson 1988, Sternberg and Hamilton, 1991) and the FRT sequences from S. cerevisiae (Broach and Hicks, 1980, Golic and Lindquist, 1989; O""Gorman et al, 1991).
A xe2x80x9csuicide genexe2x80x9d is a nucleic acid sequence which leads to cell death by transcription and possibly expression. In the vector according to the invention, it is present as part of a transcription cassette, i.e. in combination with a promoter and optionally a polyadenylation sequence.
Under one aspect of the present invention it is intended that the suicide gene is partly or completely complementary to an essential gene. The mRNA of the suicide gene produced by transcription is capable of hybridizing with the mRNA of the essential cellular gene. Consequences of the mRNA hybridization are translation arrest of the essential cellular gene and/or RNase-H activation with subsequent cell death. The essential cellular gene can be selected from cyclins and anti-apoptotic proteins, such as Bcl-2.
A further mechanism of action of the suicide gene as is intended according to the invention is that the suicide gene codes a cytopathogenic virus, such as Semliki forest virus, which kills the cell. Furthermore, the suicide gene may comprise a nucleic acid sequence coding for a suicide protein.
A xe2x80x9csuicide proteinxe2x80x9d is a natural or artificial polypeptide product which directly or indirectly leads to cell death. The indirect effect is e.g. based on the interaction with a non-toxic agent precursor so that the precursor is converted into a toxic agent which is able to trigger the death of a cell. The suicide protein may be an antigen, such as influenza hemagglutinin or foreign MHC antigens. The direct effect is here based on the stimulation of an immune response directed against the tumor cell.
With the vectors according to the invention it is possible for the first time to eliminate tumor cells with a defective transcription factor in a targeted and direct manner. The basis for the selectivity of the vector according to the invention is that the recombinase coded by the first transcription factor can only be expressed in cells with an intact transcription factor. In tumor cells with a defective transcription factor there is no binding of the transcription factor to the transcription factor binding site associated with the minimal promoter. Since the minimal promoter requires activation by the transcription factor, there is no transcription and thus also no expression of the gene encoded by the first transcription cassette, i.e. the recombinase. In contrast thereto, the coding sequence of the second transcription cassette, i.e. the suicide gene, can be transcribed because the promoter which is functionally linked to said gene is independent of any activation by a transcription factor. After having come into contact with an agent precursor, the suicide protein now expressed will metabolize said precursor, thereby contributing to the production of a toxic agent. The metabolism product will subsequently cause the death of the respectively affected cell. Alternatively, a cell type-specific antisense RNA or a virus could lead to cell death.
By contrast, in healthy cells, i.e. in cells with an intact transcription factor, the factor can bind to the transcription factor binding site so that the protein coded by the first transcription cassette, i.e. the recombinase, is expressed. On account of the recombinase target sequences which are e.g. a priori provided by the vector and which flank the region of the transcription cassette, the region located between the target sequences with the first and second transcription cassettes is immediately deleted. In the case of retroviral vectors the LTR and thus the recombinase target sequence(s) are doubled by the integration. The region located between the target sequences is also deleted. What is left in the case of a healthy cell is just a copy of the target sequence, optionally together with the LTR per previous integration site of the vector.
The transcription factor as mentioned may be any transcription factor with a defect effecting a restriction or complete elimination of the transactivator capacity. A known example of a transcription factor whose DNA binding domain in tumor cells is relatively often changed in such a way that DNA binding and thus transactivation no longer take place is the said factor p53.
In human tumors the p53 mutations are always found in the DNA binding domain. This often leads to a loss in the DNA binding capacity and thus the transactivator function of the protein. p53-binding consensus sequences were found in a number of promoters and characterized. Moreover, p53 regulates, inter alia, the expression of p21 (WAF1) (el-Deiry et al. 1994), bax (Miyashita and Reed, 1995) and IGF-BP3 (Buckbinder et al. 1995). Such a p53-binding consensus sequence is e.g. the PG motif (CCTGCCTGGACTTGCCTG) (el-Deiry et al, 1992). The inventors and others have shown that said motif (PGn) in connection with a minimal promoter (MP) makes the expression of a reporter gene p53-inducible. Such an induction is not possible with mutated p53. This has inter alia been demonstrated for the combinations PGn-CMVMin-CAT (chloramphenicol acetyl transferase) (Kern et al., 1992) and pg13-SV40Min-SEAP (secreted alkaline phosphatase). The p53-binding consensus sequence pg13 is preferred for the purposes of the present invention.
In one embodiment of the invention the flanking sequences consist essentially of the recombinase target sequences. These may be natural or synthetic sequences which are recognized by a natural or recombinant recombinase.
In a preferred embodiment the vector of the invention is derived from a retrovirus. Retroviruses are RNA viruses whose replication involves a DNA intermediate. The viral RNA genome is flanked by short repeated sequences (repeats, R) and non-repeated sequences (unique sequences, U5 and U3) which control the DNA synthesis, the integration of the virus genome in the host genome, the transcription and the RNA processing. These control regions have provided thereinbetween coding sequences for the most important structural proteins of the virus particle, namely gag and env, and for further enzymes packaged in the particle (pol, protease, reverse transcriptase and integrase). Shortly after infection the viral RNA is translated by the reverse transcriptase into DNA. Prior to integration the terminal sequences of the viral genome are doubled so that the retroviral genome is flanked by long terminal repeated sequences (long terminal repeats, LTR) which contain each a U3 region, R and a U5 region. The linear molecules are then integrated into the genome.
After integration of the reversely transcribed virus genome into the host genome, one talks about a provirus. The provirus is replicated together with the cellular host DNA and transcribed like a cellular gene. The provirus transcription is controlled by promoter and enhancer sequences which are in the U3 region of the 5xe2x80x2 LTR. Polyadenylated transcripts begin at the transition between U3 and R in the 5xe2x80x2 LTR and end in the R of the 3xe2x80x2 LTR which contains the polyadenylation signal.
Provided that specific control sequences remain within the LTRS, the retroviral genome can be exchanged for foreign DNA without impairing its ability to replicate in cells which express the proteins required for reverse transcription, integration and particle formation. To provide the enzymes required for the replication machinery, the vector DNA is transfected in cell lines which contain either complete retroviral genomes or helper viruses. The helper viruses cannot aggregate in particles due to a deletion between U5 and gag. As a consequence, only recombinant transcripts are packaged and released from the cells in virus particles. On account of said technical possibilities, retroviral vectors are preferred as vectors for gene therapy.
In the case of a vector derived from retroviruses, the 5xe2x80x2- and/or 3xe2x80x2-flanking sequences comprise complete or partial LTR regions. The at least one recombinase target sequence is here preferably in the U3 or U5 region of the 5xe2x80x2 and/or 3xe2x80x2 LTR. If the recombinase target sequence is outside the LTR, at least 2 recombinase target sequences are required. The transcription cassettes, however, are preferably outside the LTR. In the case of non-retroviral vectors at least two recombinase target sequences are provided.
The first transcription cassette contains, inter alia, a sequence coding for a recombinase. It is preferably arranged in the same transcription direction as the possibly underlying virus genome.
Any natural or synthetic protein with recombinase activity can be used as recombinase. In a preferred embodiment the recombinase encoded by the first transcription cassette is the recombinase Cre. The Cre recombinase was originally identified in coliphage P1 and is very well characterized. In preferred embodiments the recombinase target sequence for the recombinase Cre is the loxP sequence. Artificial recombinase target sequences which are recognized by the recombinase Cre can also be used. In an alternative embodiment Flp is used as recombinase. Flp derives originally from S. cerevisiae. It preferably recognizes FRT sequences from S. cerevisiae. These target sequences, in turn, can be replaced by artificial target sequences which are recognized by the recombinase Flp.
Every promoter depending on a transcription factor can be used as the minimal promoter. Preferred embodiments use xcex94CMV (Gossen and Bujard, 1992) or xcex94MMTV (Hoffmann et al., 1997). These promoters are truncated cytomegalovirus or mouse mammary tumor virus promoters which depend on a transactivation by transcription factors.
The suicide gene coded by the second transcription cassette is a thymidine kinase gene. According to the invention thymidine kinase from herpes simplex virus (HSV-TK) is particularly preferred. HSV-TK converts, for instance, added ganciclovir into a toxic metabolism product. However, all proteins that are known to produce a toxic agent from a non-toxic precursor agent after metabolization can be used as suicide proteins. A further example of such a suicide protein is cytosine deaminase.
In a further preferred embodiment the suicide gene is partly or completely complementary to an essential cellular gene. The mRNA produced by transcription of the suicide gene is capable of hybridizing with the mRNA of the essential cellular gene (antisense mechanism). Results of the mRNA hybridization are translation arrest and/or RNase-H activation which prevents the expression of the essential cellular gene and leads to cell death. Essential cellular genes, e.g., of the primary metabolism, are known to a person skilled in the art.
In a further preferred embodiment the suicide gene is able to produce a cytopathogenic virus by expression. Cytopathogenic viruses known to the person skilled in the art produce degenerative changes in the infected cells, for instance formation of giant cells, syncytia, inclusion bodies, vacuoles and granules, changes in the nucleus and lysis of the cells. An exemplary cytopathogenic virus is the Semliki forest virus.
The second transcription cassette is preferably arranged in the same transcription direction as the virus genome and the first transcription cassette; the first transcription cassette may here be located 5xe2x80x2 or 3xe2x80x2 relative to the second transcription cassette. In the case of an opposite arrangement, which is also possible, a further polyadenylation sequence must be provided for the second transcription cassette.
Any desired strong eukaryotic promoter may be used as promoter for the suicide gene. Preferred promoters are e.g. the LTR promoter of the Moloney mouse leukemia virus (MoMuLV). The person skilled in the art is aware of further promoters.
Moreover, in a further preferred embodiment the vectors according to the invention contain a selection marker cassette. Selection marker cassettes can e.g. be inserted into a U3 region. Said region can be used as evidence of the integration performed. A selection marker cassette illustrated in the examples is SV40Puro. Every other selection marker system that is transcribed and translated in eukaryotes and known to the person skilled in the art can however be used as well.
Additionally, the vectors according to the present invention can comprise any combination of transcription cassette, recombinase, promoter, transcription factor binding site, suicide gene, 5xe2x80x2 flanking sequence, 3xe2x80x2 flanking sequence, recombinase target sequence, and selection marker cassette described herein. Also, the present invention includes each embodiment of the vector described in PCT/EP99/03607 (WO 00/06758) which is hereby incorporated in its entirety by this reference.
Furthermore, the invention relates to pharmaceutical compositions which contain the vector according to the description herein and the claims. These pharmaceutical compositions are useful for the therapy of tumors which are characterized by the deficiency of a transcription factor. In preferred embodiments the vector in the therapeutic composition is packaged in a virus particle.
The vectors according to the invention are preferably replication-competent in pharmaceutical compositions. They are particularly preferably infectious, i.e. they are able to infect further cells.
The vector according to the invention can be used for treating tumor patients. In a preferred embodiment the intention is to kill tumor cells in a targeted manner by administration of the vector of the invention and subsequent administration of a precursor substance, such as ganciclovir, which is metabolizable by the suicide protein.
A further subject matter of the invention is a transduced eukaryotic cell which can be obtained by infecting a tumor cell comprising a deficient transcription factor, with a recombinant vector according to the description herein and the claims.
The following figures and examples will explain the invention: